The fibroblast growth factor (FGF) family contains eight members (Basilico et al., 1992, The FGF family of growth factors and oncogenes. Adv. Cancer Res. 59:115-165). This reference and all references cited herein are expressly incorporated herein by reference. The two prototypic members, acidic FGF (Jaye et al., 1986, Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosomal localization. Science 23:541-545; Wang et al., 1989, Cloning of the gene coding for human class I heparin-binding growth factor and its expression in fetal tissue. Mol. Cell. Biol 9:2387-2395) and basic FGF (Abraham et al., 1986, Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J. 5:2523-2528) have no signal peptide but the remaining six members, FGF-3 (Dickson et al., 1987, Potential oncogene product related to growth factors. Nature 326-833; Acland et al., 1990, Subcellular fate of int-2 oncoprotein is determined by choice of initiation codon. Nature 343:662-665), FGF-4 (Delli-Bovi et al., 1987, An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell 50:729-737; Taira et al., 1987, cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity. Proc. Natl. Acad. Sci. USA 84:2980-2984), FGF-5 (Zhan et al., 1988, The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors. Mol. Cell. Biol. 8:3487-3495), FGF-6 (deLapeyriere et al., 1990, Structure, chromosome mapping, and expression of the murine FGF-6 gene. Oncogene 5:823-831), keratinocyte growth factor (KGF) (Finch et al., 1989, Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 245:752-755) and androgen-induced growth factor (AIGF) (Tanaka et al., 1992, Cloning and characterization of an androgen-induced growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells. Proc. Natl. Acad. Sci. USA 89:8928-8932) all have signal peptides. Various members of the FGF family are involved in cell growth, differentiation, and survival as well as embryonic induction and angiogenesis (Basilico et al., supra). Because the release of aFGF and bFGF is thought to be through dead or dying cells, it is implied that they are also involved in tissue repair. Moreover, KGF mRNA has been shown to be induced more than 160 fold during wound healing (Werner et al., 1992, Large induction of keratinocyte growth factor expression in the dermis during wound healing. Proc. Natl. Acad. Sci. USA 89:6896-6900).
As with most polypeptide growth factors, the FGF signal is transduced via membrane-spanning protein tyrosine kinase (PTK) receptors (Johnson et al., 1993, Structural and functional diversity in the FGF receptor multigene family, Adv. Cancer Res. 60:1-41). The four members of the FGF receptor family, flg/FGFR1 (Ruta et al., 1989, Receptor for acidic fibroblast growth factor is related to the tyrosine kinase encoded by the fms-like gene (FLG). Proc. Natl. Acad. Sci. USA 86:8722-8726; Dionne et al., 1990, Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factor. EMBO J. 9:2685-2692; Johnson et al., 1990, Diverse forms of a receptor for acidic and basic fibroblast growth factors. Mol. Cell. Biol. 10:4728-4736), bek/FGFR2 (Kornbluth et al., 1988, Novel tyrosine kinase identified by phosphotyrosine antibody screening of cDNA libraries. Mol. Cell. Biol. 8:5541-5544; Dionne et al., 1990, supra), FGFR2 (Keegan et al., 1991, Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3. Proc. Natl. Acad. Sci. USA 88:1095-1099), and FGFR4 (Partanen et al., 1991, FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern. EMBO J. 10:1347-1354) all contain three immunoglobulin (lg)-like extracellular domains (Williams et al, 1988, The immunoglobulin superfamily-domains for cell surface recognition. Ann. Rev. Immunol. 6:381-405). The first Ig-like domain may or may not be present due to alternative splicing, resulting in either a two or three loop variant (Mansukhani et al., 1990, A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF. Proc. Natl. Acad. Sci. USA 87:4378-4382; Fujita et al., 1991, The expression of two isoforms of the human fibroblast growth factor receptor (flg) is directed by alternative splicing. Biochem. Biophys. Res. Comm. 174:946-951). This first loop has no effect on ligand binding and its function remains unknown (Johnson et al., 1990, supra; Mansukhani et al., 1990, supra). The genes of FGFR1 and FGFR2 contain three consecutive yet mutually exclusive exons that encode the 3' half of the last Ig-like domain (Champion-Arnaud et al., 1991, Multiple mRNAs code for proteins related to the BEK fibroblast growth factor receptor. Oncogene 6:979-987; Eisemann et al., 1991, Alternative splicing generates at least five different isoforms of the human basic-FGF receptor. Oncogene 6:1195-1202; Johnson et al., 1991, The human fibroblast growth factor receptor genes: A common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain. Mol. Cell. biol. 11:4627-4634; Yayon et al., 1992, A confined variable region confers ligand specificity on fibroblast growth factor receptors: Implications for the origin of the immunoglobulin fold. EMBO J. 11:1885-1890). Alternative splicing in this region generates secreted forms of these receptors and receptors with differences in their FGF binding specifities. Splicing of the first of the three exons (IIIa) into the mRNA results in a secreted form of the receptor containing no transmembrane or PTK domain (Johnson et al., supra., 1990, 1991). If the next exon (IIIb) is spliced into the mRNA, a membrane spanning PTK receptor with a high affinity for aFGF and KGF results. When considering FGFR2, this isoform is referred to as the KGF receptor (Miki et al., 1991, Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science 251:72-75; 1992, Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene. Proc. Natl. Acad. Sci. USA 89:246-250; Yayon et al., 1992, supra). Inclusion of the last of these three exons (IIIc) confers high affinity to aFGF, bFGF and FGF-4 (Dionne et al., 1990, supra; Mansukhani et al., 1992, Characterization of the murine BEK fibroblast growth factor (FGF) receptor: Activation by three members of the FGF family and requirement for heparin. Proc. Natl. Acad. Sci. USA 89:3305-3309) but not to KGF (Miki et al., 1992, supra). This FGFR2 isoform is referred to as a "bek-like" receptor.
Expression patterns of several FGF proteins during development are well documented (Whitman et al., 1989, Growth factors in early embryogenesis. Annu. Rev. Cell Biol. 5:93-117; Hebert et al., 1990, Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis. Dev. Biol. 138:454-463; Niswander et al., 1992, FGF-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114:755-768; Tannahill et al., 1992, Development expression of the Xenopus int-2(FGF-3) gene: Activation by mesodermal and neural induction. Development 115:696-702). The FGFs have also been implicated in amphibian limb regeneration but their specific role in this developmental process remains obscure. When FGF is infused into the distal stump of denervated newt limbs, cell cycling is stimulated over the depressed level normally seen after denervation (Mescher et al, 1979, Mitogenic effect of a growth factor derived from myelin on denervated regenerates of newt forelimbs. J. Exp. Zool. 207:497-503; Gospodarowicz et al., 1980, Fibroblast growth factor and the control of vertebrate regeneration and repair. Ann. N. Y. Acad. Sci. 339:151-174). By binding assays and Western blotting analysis, (Boilly et al., 1991, Acidic fibroblast growth factor is present in regenerating limb blastemas of axolotis and binds specifically to blastema tissue. Dev. Biol. 145:302-310), showed that aFGF and its receptor(s) are present within the newt limb blastema; nevertheless, the cellular source of this growth factor was not determined. In the mouse limb bud, FGFR2 transcripts were detected in the surface ectoderm, whereas FGFR1 transcripts were distributed diffusely in the mesenchyme (Orr-Urtreger et al., 1991, Developmental expression of two murine fibroblast growth factor receptors, fig and bek. Development 113:1419-1434; Peters et al., 1992, Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development 114:233-243).
According to the present invention cDNAs of newt aFGF was isolated and characterized.
According to the present invention, cDNAs of newt FGFR1 and FGFR2, FGFR3 and KGFR were cloned. Riboprobes made from these cDNAs were used to carry out in situ hybridization at various stages of newt limb regeneration.
Further according to the present invention there is disclosed cells transfected with a DNA sequence encoding human acidic fibroblast growth factor and capable of expressing said factor.
An object of the present invention is to provide purified and synthetic forms of newt aFGF.
An additional object of the present invention is the determination of the amino acid sequence of such aFGF.
A further object of the present invention includes providing purified forms of newt aFGF and mammalian cell lines expressing FGF receptors which would be valuable to evaluate agonist and antagonist proteins such as human FGF proteins.